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The COP9 signalosome (CSN) is an evolutionary conserved multiprotein complex that regulates many aspects of plant development by controlling the activity of CULLIN-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate and target for proteasomal degradation a vast number of specific substrate proteins involved in many developmental and physiological processes, including light and hormone signaling and cell division. As a consequence of CSN pleiotropic function, complete loss of CSN activity results in seedling lethality. Therefore, a detailed analysis of CSN physiological functions in adult Arabidopsis plants has been hampered by the early seedling lethality of csn null mutants. Here we report the identification and characterization of a viable allele of the Arabidopsis COP9 signalosome subunit 4 (CSN4). The allele, designated csn4-2035, suppresses the adventitious root (AR) phenotype of the Arabidopsis superroot2-1 mutant, potentially by altering its auxin signaling. Furthermore, we show that although the csn4-2035 mutation affects primary and lateral root (LR) formation in the 2035 suppressor mutant, CSN4 and other subunits of the COP9 complex seem to differentially control AR and LR development.

The plant hormone auxin plays a central role in adventitious rooting and is routinely used with many economically important, vegetatively propagated plant species to promote adventitious root initiation and development on cuttings. Nevertheless the molecular mechanisms through which it acts are only starting to emerge. The Arabidopsis superroot2-1 (sur2-1) mutant overproduces auxin and, as a consequence, develops excessive adventitious roots in the hypocotyl. In order to increase the knowledge of adventitious rooting and of auxin signalling pathways and crosstalk, this study performed a screen for suppressors of superroot2-1 phenotype. These suppressors provide a new resource for discovery of genetic players involved in auxin signalling pathways or at the crosstalk of auxin and other hormones or environmental signals. This study reports the identification and characterization of 26 sur2-1 suppressor mutants, several of which were identified as mutations in candidate genes involved in either auxin biosynthesis or signalling. In addition to confirming the role of auxin as a central regulator of adventitious rooting, superroot2 suppressors indicated possible crosstalk with ethylene signalling in this process.

Map-based cloning (MBC) is the conventional approach for linking phenotypes to genotypes, and has been successfully used to identify causal mutations in diverse organisms. Next-generation sequencing (NGS) technologies offer unprecedented possibilities to sequence the entire genomes of organisms, thereby in principle enabling direct identification of causal mutations without mapping. However, although mapping-by-sequencing has proven to be a cost effective alternative to classical MBC in particular situations, methods based solely on NGS still have limitations and need to be refined. Aiming to identify the causal mutations in suppressors of Arabidopsis thaliana superroot2 phenotype, generated by ethyl methane sulfonate (EMS) treatment, we combined NGS and classical mapping, to rapidly identify the point mutations and restrict the number of testable candidates by defining the chromosomal intervals containing the causal mutations, respectively. The NGS-assisted mapping approach we describe here facilitates unbiased identification of virtually any causal EMS-generated mutation by overlapping the identification (deep sequencing) and validation (mapping) steps. To exemplify the useful marriage of the two approaches we discuss the strategy used to identify a new viable recessive allele of the Arabidopsis CULLIN1 gene in the non-reference Wassilewskija (Ws-4) accession.

The development of shoot-borne roots, or adventitious roots, is indispensable for mass propagation of elite genotypes. It is a complex genetic trait with a high phenotypic plasticity due to multiple endogenous and environmental regulatory factors. We demonstrate here that a subtle balance of activator and repressor AUXIN RESPONSE FACTOR (ARF) transcripts controls adventitious root initiation. Moreover, microRNA activity appears to be required for fine-tuning of this process. Thus, ARF17, a target of miR160, is a negative regulator, and ARF6 and ARF8, targets of miR167, are positive regulators of adventitious rooting. The three ARFs display overlapping expression domains, interact genetically, and regulate each other's expression at both transcriptional and posttranscriptional levels by modulating miR160 and miR167 availability. This complex regulatory network includes an unexpected feedback regulation of microRNA homeostasis by direct and nondirect target transcription factors. These results provide evidence of microRNA control of phenotypic variability and are a significant step forward in understanding the molecular mechanisms regulating adventitious rooting.